From Proteopedia

Revision as of 09:14, 30 October 2010

Lysozyme

Lysozyme is an important enzyme that is commonly found in vertebrate cells and secretions capable of destroying cell walls.

Activity

Lysozyme is an enzyme effective in its catalytic degradation of the peptidoglycan in bacterial cell walls, specifically by catalyzing the hydrolysis of 1,4 beta linkages of the residues, N-acetylmuramic acid and N-acetyl-D-glucosamine in cell wall peptidoglycan.Image:Lysozyme-B.jpg

This enzyme similarly catalyzes hydrolysis of 1,4 beta linkages of poly N-acetylglucosamine residues of chitin, a major constituent of fungi cell walls as well as exoskeletons such as those of insects and crustaceans ^[1].

Occurrance

This cell wall degradation often leads to cell death, displaying the bactericidal function of lysozyme. The effectiveness of lysozyme in degrading cells walls, especially those of gram-positive bacterial cells is a function that naturally leads to the incorporation of the lysozyme enzyme into organisms as a bactericidal agent. Lysozyme is readily present in the living cells and vertebrate mucosal secretions including tears and saliva^[2].

History

Initially in 1909 Laschtschenko described lysozyme which he found displayed antibacterial effects in chicken eggs. Alexander Fleming later observed the antibacterial lysing action of lysozyme by treating cells with mucus, and developed the name lysozyme. David Phillips solved the structure of lysozyme using X-ray diffraction methods. In order to determine the mechanism of an enzyme, one must first assess the enzyme substrate complex. Three dimensional arrangement of the enzyme around the substrate in the enzyme's active site is difficult to asses because of the brief binding period to the substrate before the reaction. Phillips was able to utilize a trisaccharide as substrate in place of the preferred six saccharide substrate, which allowed for X ray diffraction due to the slow hydrolization process. Phillips then used model building to determine the binding of lysozyme to the six membered oligosaccharide substrate. Lysozyme was only the second protein and the first enzyme to have been structurally determined using these X ray diffraction procedures. Phillips determined the full sequence of lysozyme as well as the enzyme structure, and deduced the mechanism performing the catalytic process of lysozyme. This mechanism provided insight into the influence of structure on the catalyzing action of lysozyme and enzymes as a whole ^[3].

Structure

The presence of lysozyme in hen egg whites has led to a particular lysozyme, Hen egg white (HEW) lysozyme, which prevails as the most heavily studied lysozyme species. This increased understanding of the the lysozyme mechanism from the extensive studies of HEW lysozyme, allows a more specific analysis of lysozyme structure and specific function. Visualize the overall structure of .

Hen egg white lysozyme is a smaller ellipsoidal enzyme composed of 129 amino acid residues leading to a single polypeptide chain of 14.3 kD. The amino acid polypeptide sequence of 129 amino acid residues is responsible for the secondary and tertiary structure of the lysozyme molecule. This polypeptide backbone can be mapped from amino to carboxyl end, displaying the path of the polypeptide after proper folding has occurred. The amino terminus to the carboxy terminus of the polypeptide chain can be traced of lysozyme from blue (amino) to red (carboxy) in the ribbon representation of ^[4].

The backbone of this 129 amino acid enzyme conforms to a very specific and ordered molecule with the help of the secondary structural interactions. Lysozyme contains five regions and five regions containing as displayed in this . Linking these secondary structures, a number of beta turns and large amount of random coil makes up the remainder of the polypeptide backbone. The polypeptide backbone of lysozyme involved in the 3 antiparallel beta sheets display the beta hairpin motif of supersecondary structure. The increased stability of the antiparallel beta sheet due to the proper alignment of hydrogen bonds between sheets allows for the presence of the increased stability found in the Beta hairpin motif ^[5].

Hydrogen Bonding

The hydrogen bonding patterns in secondary structure have an important role in determining protein structure. Limited by the various torsion angles of the alpha carbon-nitrogen and alpha carbon-carbon bonds in each residue, each secondary structure (alpha helices and beta sheets) display specific patterns of hydrogen bonding of the amino acid residues. The yellow lines outline the various interactions between the various hydrogen bond donators and acceptors in this lysozyme molecule.

Disulfide Bonding

The conformation of the lysozyme peptide is affected by the presence of 4 disulfide bridges between 8 Cystein residues occurring within the peptide backbone. The presence of these disulfide bridges assist in the folding, stability, and overall function of lysozyme. The are colored yellow, and the 8 participating Cystein residues are colored green^[6]. .

The amino acids present in the lysozyme polypeptide sequence have a direct influence not only on primary structure, but also on the secondary structural changes as well as the tertiary structural changes which can be influence by polarity and charge of the sidechains. The various amino acid differ in their properties because of the great variety of side chains present on each amino acid. Polar and nonpolar, and charged and uncharged side chains lead to various degrees of hydrophobicity and hydrophilicity which can have a very dominant effect on protein folding. In lysozyme, these are displayed for each amino acid residue.

Polarity

The nature of the amino acid sidechains in the lysozyme polypeptide sequence leads to regions of varying hydrophobic natures and polarities of the enzyme structure. The presence of certain regions of hydrophilicity and hydrophobicity is a driving force in determining protein structure when folding. The varying polarities of the side chains influence the locations of residues in the enzyme structure. Nonpolar residues appear blue, and Polar residues appear red in the following display of lysozyme. Nonpolar residues will display hydrophobic tendencies occurring mostly on the interior of the enzyme while polar residues will increase in abundance on the surface of the protein in order to increase contact with the aqueous solvent satisfying their hydrophilic nature. By observing a spacefilled structural depiction of with polar molecules colored red and nonpolar molecules colored blue the influence of polarity on nucleotide arrangement and protein folding is evident, with the blue (nonpolar) regions inside the red (polar) regions. The presence of interacting with thee various hydrophilic residues is depicted to further display how polarity affects structure. Water is depicted as yellow, and the polar and nonpolar regions remain their respective color.

Charge

Charges of the various regions of the lysozyme structure display a hydrophilic nature and thus also affect the location of that region of polypeptides and overall folding of the protein. Charged regions of the protein will display hydrophilic tendencies and therefore will most often be located on the surface of the lysozyme molecule where they can interact with the aqueous solvent. Non-charged portions will display hydrophobic tendencies and be located on the interior of the molecule. The effect of various on protein structure can be visualized with charged molecules represented by red anionic and blue cationic regions, and uncharged regions colored in grey. This depiction of lysozyme uses a spacefill representation of lysozyme to depict .

Active Site

Enzymes contain a mechanism of action that utilizes the specificity of binding to a substrate to produce an enzyme substrate complex, which quickly proceeds with the mechanism. The active site has specific residues that facilitate the binding of the substrate through a specific three-dimensional arrangement, prior to catalyzing the reaction and releasing products. Lysozyme contains a cleft in its structural arrangement, which allows for the location of the substrate-binding site. The active site of lysozyme is arranged to accommodate an oligosaccharide substrate size of six residues^[7].

Residues

Along with this geometric complementing of the active site, the sidechains of Glu 35(green) and Asp 52 (yellow), which are considered lysozyme’s , are folded to the appropriate location helpful in assisting in the catalysis of the glycosidic bond hydrolysis. Specifically, the transiently formed oxonium ion produced by the protination of the oxygen atom and the subsequent cleavage of the C-O bond requires stabilization by the actions of these two active site residues. Lysozyme mediates this reaction, and the assistance of the general acid base catalysis of Glu 35 and the electrostatic catalysis of the Asp 52 residue is necessary in order to stabilize the ion transition state and accomplish the bond cleavage and regeneration of the active site groups to yield a product.

Ligand

The presence of a ligand on the lysozyme molecule would display a smaller molecule bound to the surface of the lysozyme molecule. The lysozyme molecule is represented in grey, and the in red. Ligands can biologically assist in the catalysis of reactions. Enzyme substrates can be viewed as ligands that directly bind to the active site of the enzyme. In the case of the lysozyme mechanism, the oligosaccharide consisting of 6 substrate residues would bind to the active site of the lysozyme molecule to begin the reaction. The presence of a ligand clearly distinguished from the enzyme components is visible in this model ^[9].

Mechanism

Lysozyme catalyzes a reaction, specifically the hydrolysis of a glycoside and conversion of an acetal to a hemiacetal. Lysozyme will attach to a bacterial cell wall through the binding to the hexasaccharide substrate. The D residue of this oligosaccharide in order to properly fit in the active site must be distorted to the half chair conformation. Following the transfer of a Glu 35 proton to the O1 atom between the D and E sugar rings, the cleavage of the C1-O1 bond forms the positively charged oxonium ion. The formation of the oxonium ion is encouraged by the strain from the half chair distortion of the D ring, as catalysis through preferential binding of the transition state. The Asp 52 carboxylate group performs electrostatic catalysis to stabilize the positive charge of this transition state. The nucleophilic attack of the Asp 52 carboxylate group to the C1 of the D ring forms a covalent intermediate. Water replaces the E sugar ring assisting the catalytic cycles process. Glu 35 performs general base catalysis to assist in hydrolyzing the covalent bond, forming another oxonium ion transition state. The reaction is complete when lysozyme releases the D ring product and the active site residues regenerated^[10].

Figure

The mechanism proceeds as follows:

Image:Lysozyme-mech.gif^[11]. Glu 35 acts as a general acid catalyst and a general base catalyst, and Asp 52 acts as a covalent catalyst, to help mediate the reaction with a significant increase in rate of hydrolysis of the substrate than in an uncatalyzed equivalent reaction.

Lysozyme Applications

Lysozyme and the efficient antibacterial properties that it displays contain numerous applications in many different fields. Lysozyme is often used in molecular labs in alkaline lysis procedures for the isolation of plasmid DNA. Pharmaceutical uses of lysozyme provide a highly efficient antibacterial agent that can destroy gram-positive microorganisms responsible for a variety of infections. Lysozyme is capable of supporting our already existing defenses against foreign bacterial contaminants that can lead to sickness. The highly “lytic activity” of lysozyme on bacterial cell walls in general leads to the ability of food industry applications of lysozyme in preventing food spoilage. The decontaminating applications of lysozyme span a wide variety of industries, a display of the true power and efficiency of lysozyme^[12].

References

1. ↑ Catalysis by Lysozyme. Functuion of Lysozyme. Lysozyme reaction. Enzyme (EC 3.2.1.17). (n.d.). Lysozyme. Retrieved October 30, 2010, from http://lysozyme.co.uk/lysozyme-enzyme.php
2. ↑ Lysozyme. (n.d.). Lysozyme. Retrieved October 30, 2010, from http://lysozyme.co.uk/
3. ↑ Weaver, R. F. (2007). Molecular Biology (4 ed.). New York: McGraw-Hill Science/Engineering/Math
4. ↑ FirstGlance in Jmol. (n.d.). molvis.sdsc.edu. Retrieved October 30, 2010, from http://molvis.sdsc.edu/fgij/
5. ↑ Protein Data Bank. (n.d.). PDB Data Bank. Retrieved October 30, 2010, from www.rcsb.org/pdb/results/results.do?outformat=&qrid=B1D4D57E&tabtoshow=Current
6. ↑ Disulfide Bonds. (n.d.). Bridgewater State University - Personal Home Pages. Retrieved October 30, 2010, from http://webhost.bridgew.edu/fgorga/proteins/disulfide.htm
7. ↑ Lysozyme - Worthington Enzyme Manual . (n.d.). Enzymes, Biochemicals: Worthington Biochemical Corporation . Retrieved October 30, 2010, from http://www.worthington-biochem.com/LY/default.html
8. ↑ Pratt, C. W., Voet, D., & Voet, J. G. (2008). Fundamentals of Biochemistry: Life at the Molecular Level (3 ed.). New York, NY: Wiley.
9. ↑ Quantitative determination of lysozyme-ligand binding. (n.d.). Pubmed Central. Retrieved October 30, 2010, from ukpmc.ac.uk/abstract/MED/17922488;jsessionid=B7C92
10. ↑ Chippman, D. (2001). Mechanism of Lysozyme Action. Science Magazine, 165(3892), 454-465. Retrieved October 29, 2010, from the Sciencemag database.
11. ↑ Lysozyme. (n.d.). Victoria University of Wellington - Home Page . Retrieved October 30, 2010, from http://www.vuw.ac.nz/staff/paul_teesdale-spittle/essentials/chapter-6/proteins/lysozyme.htm
12. ↑ Lysozyme. (n.d.). Chemicals & Health supplements | nutritional & herbal supplements, vitamins, pharmaceuticals, amino acids. Retrieved October 30, 2010, from http://www.greatvistachemicals.com/proteins-sugars-nucleotides/lysozyme.html